Calcitonin originated from platypus and application thereof
A technology of calcitonin and platypus, which is applied in the field of biomedicine, can solve the problems of heavy burden of medical expenses for patients, limited calcitonin, and large clinical dosage, so as to reduce clinical dosage, lower serum calcium content, and significantly reduce The effect of blood calcium bioactivity
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Embodiment 1
[0025] Example 1: Obtain calcitonin sequence by BLAST and construct phylogenetic tree
[0026] Using the salmon calcitonin sequence (CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP) as the query sequence, the blastp program of BLAST was used to search the non-redundant protein database in the NCBI database, and the large yellow croaker, hippocampus, grouper, three species of golden-thread barb, Arowana and two A local BLAST comparison of the genomes of aquatic organisms such as Mudskipper and a total of 68 protein sequences from different species was obtained, 13 of which were from aquatic organisms.
[0027] Further analysis of the sequences found that there were many repetitions in the amino acid sequences of the alignment segments corresponding to 32 amino acids of salmon calcitonin among the 68 protein sequences. 27 different calcitonin polypeptide sequences (SEQ ID NO.1-SEQ ID NO.27) including the calcitonin sequence.
[0028] Then, utilize the neighbor-joining (N-J) method of MEGA 5 (...
Embodiment 2
[0034] Example 2: Solid phase chemical synthesis of calcitonin polypeptide
[0035] The structure of platypus-derived calcitonin is as follows:
[0036] Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Asn-Pro-Arg-Thr- Asp-Val-Gly-Ala-Gly-Thr-Pro-NH 2 .
[0037] In the above amino acid sequence, Pro-NH2 represents prolinamide, and the two Cys residues at positions 1 and 7 are connected by a disulfide bond.
[0038] The 27 calcitonin polypeptides involved in Example 1 of the present invention were synthesized by a solid-phase or liquid-phase method. In this example, the synthesis of platypus-derived calcitonin by Fmoc (9-fluorenylmethoxycarbonyl) solid-phase synthesis method is used as an example to illustrate the polypeptide synthesis process, but it is not limited to this method for polypeptide synthesis. The steps are as follows:
[0039] (1) Select (2,4-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHA resin (i.e. Rink ...
Embodiment 3
[0046] Example 3: Purification and Mass Spectrometry Identification of Calcitonin Polypeptide by High Performance Liquid Chromatography (HPLC)
[0047] The platypus-derived calcitonin polypeptide obtained in Example 2 was purified by reverse-phase high-performance liquid chromatography (RP-HPLC). The purification conditions are as follows: the chromatographic column is a C18 reversed-phase column (Hichrom Vydac 218TPTM C-18Reversed-PhaseColumns, 5μm, 4.6mm i.d.x 250mm), mobile phase A is 0.1%TFA-100%H 2 O, mobile phase B is 0.1% TFA-100% CNCH 3 , the flow rate is 1ml / min, the linear elution gradient is from 20% to 90% of mobile phase B within 30min, and from 90% to 100% of mobile phase B within 5min.
[0048] Such as figure 2 As shown, the obtained platypus-derived calcitonin polypeptide was identified by HPLC, and its purity reached more than 96%. Such as image 3 As shown, the identification result of ESI mass spectrometry is in line with the theoretical value, the mo...
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